Top of pageAbstract Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. This approach requires a safe procedure to stably modify myogenic cells so that they express the large dystrophin transgene. We used nucleofection to introduce DNA plasmids coding for eGFP or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. This combination of methods eliminates the need for viral vectors and reduces the risk of insertional mutagenesis. Following nucleofection of a plasmid expressing eGFP, 50% of MD1 cells, a mouse muscle-derived stem cell line, and 60% of normal human primary cultured myoblasts transiently expressed the fluorescent protein. But stable expression was rare. In both cell types, co-nucleofection of a plasmid expressing the phiC31 integrase and a plasmid containing the eGFP gene carrying a 285 bp attB sequence produced 15 times more frequent stable eGFP expression, due to site-specific integration of the transgene into the genome. Co-nucleofection of the phiC31 integrase plasmid and of a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after one month of culture. The presence of eGFP-full-length dystrophin protein in myotubes was confirmed by Western blotting. Finally, MD1 stem cells expressing integrated eGFP were successfully transplanted into leg muscles of mdx mice, leading to the presence of green fluorescent fibers. A non-viral approach combining nucleofection and the phiC31 integrase may eventually permit safe auto-transplantation of genetically modified myogenic cells to muscular dystrophy patients.